Method for stimulating an immune response

ABSTRACT

A method is described whereby dendritic cells derived from the CD34+ and CD 34−hematopoietic cell lineages are directed to become programmable antigen presenting cells. The programmed cells may be pulsed with tumor cell RNA or tumor cell RNA expression products. The protocol provides for directing the maturation of dendritic cells to become antigen presenting cells. The protocol further provides for isolating tumor cell RNA from biopsy material that has been prepared in paraffin block storage. The directed dendritic cell is provided with a plurality of tumor markers by using tumor RNA in toto, the poly A+RNA fraction or the expression product of such RNA. Once activated the dendritic cells are incubated with T4 and T8 lymphocytes to stimulate and sensitize the T lymphocytes which upon introduction either into a donor host or a nondonor recipient will provide immune response protection.

FIELD OF THE INVENTION

This application is a divisional of U.S. patent application Ser. No.09/746,632, filed Dec. 20, 2000, now U.S. Pat. No. 6,645,487 issued Nov.11, 2003, which is a continuation of U.S. patent application Ser. No.09/017,842, filed Feb. 3, 1998 now U.S. Pat. No. 6,251,665, issued Jun.26, 2001, which claims the benefit of U.S. Provisional Application Nos.60/042,110 filed Mar. 26, 1997, 60/038,736 filed Feb. 14, 1997, and60/037,435 filed Feb. 7, 1997. The present invention relates generallyto stimulation of hematopoietic progenitor cells of blood and bonemarrow origin for the purpose of eliciting cell differentiation. Morespecifically, this invention relates to methodologies for specificallychanneling the differentiation process of hematopoietic stem cells intoDendritic Cells (DCs) and particularly, specifically causing thedifferentiation of the hematopoietic cells into DCs so that they becomeProgrammable Antigen Presenting Cells (pAPCs) having the capacity todirect a complete and/or specific immune response to a variety oftargeted markers encoded by tumor RNA which markers are presented to thepAPC creating a Programmed Super-Presenting Cell (PSAPC) whichsubsequently may provide a treatment for specific disease states.

BACKGROUND

In recent years there have been numerous advances in the level ofunderstanding of how cancer cells grow inside a host. Generally, it isknown that where a tumor or cancer becomes manifest, either there is adeficiency in the host's immune system and/or the tumor cells secrete orexpress agents which block the normal response of the host's immunesystem. In any event, there is a failure on the part of the host'simmune system to recognize the presence of the cancer cell as“non-self”. Because of this failure, the tumor cell and its progeny areallowed to grow without the benefit of predatory attack from the host'simmune system cells which are normally responsible for detection ofabnormal conditions. Primarily, the immune cells responsible for suchpredatory attack are the white blood cells of the CD34 lineage includingthe lymphocyte-activated killer macrophages and the T8 killer cells.Cells derived from CD34 lineage naturally become differentiated to tenor more mature cell types dedicated to specific functions. Thefunctionality is believed to be determined by factors, such ascytokines, leading to the next differentiated stage.

Although seemingly much is known of specific hematopoietic cells whichhave become differentiated into identifiable discrete cell types, littleis known about the physiologic control mechanisms involved in suchdifferentiation process. Thus, contemporary research has centeredprimarily on examination of specifically known cell types and the cellsurface “markers” recognizable at each such differentiation stage.Conspicuously lacking in the art has been clearly useful information orunderstanding of physiological events taking place within the cells asthey metamorphosized from one state to the next differentiated state.

Consistent with the current state of understanding such celldifferentiation is the methodology utilized by leading physicians andresearchers in treatment protocols for cancerous diseases. Over the pastseveral decades, cancer treatment methodologies have centered onconventional therapies such as surgical excision, radiation, and theinjection of potent chemical agents. Such methodologies have wellrecognized limitations and have, in many cases, been proved to causemuch additional pain and suffering to the patient as well as unreliablelong-term effectiveness.

Numerous recent treatments have attempted to affect tumor cells bydirect manipulation of cells understood to be active in clearing thebody of dead or improperly functioning cells. Understandably, the cellstargeted for investigation have involved cells of the immune responsesystem. However, recent attempts at blocking growth of tumor cells,though utilizing sophisticated methodologies (such as by attempting toblock the immune suppression capacity of the tumor cells) have generallybeen unsuccessful. These attempts are still ongoing and are also ofquestionable benefit in bringing about reliable treatments resulting inlong-term tumor remission.

Examples of methodologies in the recent art include targeted radiationand chemotherapy, injection of cytokines, injection of monoclonalantibodies to specific known tumor cell surface markers, and genetictherapies involving transforming cells with genes encoding factorsbelieved to affect specific tumor states. One methodology has involvedutilizing a class of natural immunostimulatory agents, particularlylymphokines, which are known to act as immunomodulators. Somelymphokines are produced by one T lymphocyte but act by signaling otherT lymphocytes. Prior attempts have been disclosed in the art to regulatesuch immunomodulators by adding factors, such as Interleukin 2 (IL-2),to enhance or elicit an immune response to tumor cells and thereby trumpthe immunosuppression effect that many tumor cells exhibit. Thedifficulty with such past investigations directed at blockingimmunosuppression is that they have either failed entirely or have onlyattacked specific antigenic markers produced by the tumor cells. Othermethods of treatment have included direct injections of variouscytokines. Still other methods have attempted stimulating the patient'simmune response cells using cytokines in the presence of the patient'sown cancer cells, then re-injecting the treated immune response cells. Anumber of attempts have been made along these lines and a significantpercentage of the patients do not respond optimally to suchinterventions.

The results of treatments utilizing any of the above methods indicatethat subpopulations of cancerous cells remain undetected and unaffectedand are able to present later clinical manifestation of the cancerousstate. For example, a number of very malignant cancers, such asglioblastoma multiforme, continue to be a death sentence prognosis forpatients who are so afflicted. Virtually all patients relapse, evenafter conventional debulking, chemotherapy and radiation therapy.Typical survival after diagnosis is usually 18 months.

Other regiments include gene therapy. For example, when TGF-β detectiongene is inserted into a host's tumor cells in vitro, then injected toattempt to elicit an immune response, treatments are only temporarilysuccessful and fail to provide a lasting benefit, even when combinedwith IL-2 co-stimulatory regimens. The temporary effect results becausenot all of the tumorous cells have been eliminated. This is becausepopulations of tumor cells are heterogeneous in the variety of surfacemarkers they present. Not all such markers will be available forpresentation to cells responding to the protective response effects ofTGF-β or IL-2. Thus, some cells are not properly recognized in thetreatment regime and survive undetected.

There is therefore an ongoing need for a means of stimulating moreeffectively and completely the host's immune response to serious diseaseand cancer states. The current invention has centered on the recognitionthat dendritic cells derived from precursor CD34+ and CD34− stem cellsmay be specifically directed to become a programmable antigen presentingcells (pAPCs). The current invention shows that in fact the pAPCs mayindeed be programmed to become programmed super antigen presenting cells(pSAPCs) having the capacity to elicit an immune response to any numberof tumor antigen moieties after being “loaded” with either tumor derivedRNA in toto or the poly A+population thereof, or with the expressedproteins encoded by such RNAs including immune significant tumorantigens expressed therefrom.

It will be well appreciated in the hemopoietic cell art that dendriticcells are typically bone marrow-derived leukocytes which are known toplay a central role in cellular immune responses. There are many aspectsof dendritic cell ontogeny which remain poorly defined. However, moststudies suggest that these dendritic cells emerge from the bone marrow,circulate in the peripheral blood in an immature form, and then entertissues where they function as antigen-presenting cells or differentiateinto macrophages. Once these dendritic cells capture a foreign body orsome type of cell recognized as non-self, they then migrate to centrallymphoid organs where they present these antigens to the T lymphocytes.Once the dendritic cell makes the presentation to the T lymphocytes, theT lymphocytes then mount an immune response.

Dendritic cells are difficult to study due to the scarcity of theirpopulations and difficulty in growing these cells in cell culture.Dendritic cells can be derived from three readily available sources: (1)peripheral blood monocytes, (2) bone marrow and (3) umbilical cordblood. The functional differences between dendritic cells which arederived from the peripheral blood monocytes and those derived from bonemarrow remain controversial. Dendritic cells possess idealcharacteristics to be used as antigen-presenting cells. The key problemsexperienced by researchers have been both the inability to retrievedendritic cells in sufficient quantity and to direct a stem cell todevelop into a dendritic cell either in sufficient quantity orsufficient specificity. Therefore, if dendritic cells could be properlypropagated and channeled, the fact dendritic cells possess idealcharacteristics to generate a tumor-specific cellular immune response byprocessing and presenting tumor-associated antigens to primed CD4+Tlymphocytes, dendritic cells would offer a highly desirable andefficient means to initiate an immune response.

Moreover, the current state of the art in cancer research has focused onthe science of recombinant DNA sequencing. In general, researchers aresearching the genomes of cells for DNA sequences encoding genesresponsible for causing either the cancerous state itself, or thecancer's immunosuppressive effects. At least 6,000 genes have beenidentified and characterized. The human genome itself is estimated toharbor at least 100,000 genes. Additionally, it is believed that anygiven cell may express 20 to 45 thousand different genes during its lifecycle, if not at one time. Cancer cells are believed to express numerousgenes in addition to, or in lieu of, those normally expressed and infact may express a greater number than the average normal cell.

Previously, researchers have focused on identifying various unique genessuch as Her2neu or Brac-a, and have associated such specific genes withspecific cancers. Unfortunately, by focusing on single genes soassociated with a cancer, the possibility that such genes may havelittle significance with respect to an immunological response greatlyincreases. The reason that such single genes may not be all important tothe cancer state and immune response is that such cancer cells areheterologous, not homologous, with respect to expression of surfaceantigen markers.

The present invention furthers the state of the art by making it clearthat it is not significant to identify every single gene that isexpressed on a cancer cells. Rather, that it is important to provide ameans by which the expressed genes of a cancer cell may be presentedalong with, or in combination to, the immune response system by a meansdirectly useful to the “natural” mechanisms of recognition utilized byimmune system cells. The inventors of the present invention delineatehow this may be accomplished by directed growth of dendritic cells to astate where they may become programmable antigen-presenting cells(pAPCs), capable of digesting a foreign cell (non-self), or ingestingforeign cell RNA in toto or as the poly A+portion thereof, or theexpression product encoded by such RNAs. The inventors intend for thepAPCs to select appropriate cancer RNA or RNA expression products to bemost appropriate for presentation.

Although a similar digestion of non-self matter occurs in the naturalsetting with the aid of macrophages and other phagocytotic cells, thepresent invention avoids the conditions understood to occur in vivo andaccomplishes enhanced digestion and presentation in vitro. The currentinvention provides for uniform conditions under which dendritic cellsmay be directed or evolved to a state where they may be highly effectivein digesting and/or selecting appropriate cancer and other cell markersfor presentation. The inventors hereby suggest that during the digestionprocess, the dendritic cell will itself identify those antigens ofsignificance for the immune system meaning that it will select out some10 to 20 or more antigens from a specific cancer cell, RNA, or RNAencoded product which have immunological significance. Under in vivoconditions of a host afflicted with a cancer, such selection may not beeffectively recognized by the immune system, especially one that iscompromised or masked. In contrast, under conditions of the currentinvention, the dendritic cell selected markers may be presented to T4and T8 cells in an environment which will allow such T4 and T8 cells tobecome properly educated and activated so as to trigger a useful immuneresponse.

The current invention provides a means by which the dendritic cell canbe activated to become a “programmable” antigen-presenting cell or pAPCwhich is a “manufactured” dendritic cell line and which can further be“immortalized”. Immortalization of the pAPC allows for a suitablecontinuous source of cells which may comprise the basis of an allogenicvaccine. Therefore, the donor host or any host of the same allotype withthe same disease, exhibiting such RNA in toto or poly A+ portionthereof, or the encoded protein therefrom, with this source of allogenicdendritic cells will create vaccines directed to particular tumors.Similarly, these allogenic dendritic cells may be mixed withrepresentative samples of different tumors' RNAs or RNA encodedproducts. For instance, the current invention contemplates combining aplurality of tumor's RNAs and/or RNA expression products fromprogressive stages of the same cancer type. By representingdifferentiation periods in the disease state progression, aheterogeneous population of tumor cell antigens is presented to the pAPCand therefore a single vaccine may be created representing “differentphases” of the cancer giving rise to a multivalent vaccine. Therefore,one vaccination using such an allogenic-based vaccine may protectagainst the whole spectrum of a specified cancer. Similarly, anallogenic vaccine for one very poorly differentiated cancer with manyatypical features may also have an effect on more early stages of thesame type of cancer or a different cancer, meaning that a vaccine madefrom allogenic pAPCs to glioblastoma tumor tissue, or its RNA, or itsRNA expression products for instance, may prove efficacious in use withother unrelated tumors such as a prostate cancer. The current inventionfurther provides for “commonalities” between all cancers which makespossible a single allogenic vaccine that is effective for a multiple ofdifferent cancers. Thus, many features which are similar to all cancersmay be provided for in a pSAPC for presentation to cells of the immunesystem, in effect providing immune-specific antigens with commonalitybetween different tissue types.

The current invention also contemplates use in placing specific antigenson the pAPC which function in a regulatory surveillance mode to preventrecurrence of a new cancer or a heterozygous group of cancer cells fromgrowing out of control. For example, a pAPC “programmed” for lung cancermay be used in effectively eliminating a host's cancer using an“autogenic” lung cancer vaccine. Where a sub-group of cells survive thisimmune response, because a heterozygous group of antigens is notrecognized as that presented by the pSAPC, a different pSAPC with“memory” to capture di novo cancers may also be used to educate immunesystem cells. Such pSAPCs equipped with “surveillance antigens” may beeffective not only against a heterozygous group of tumor antigens butmay also be used for surveillance in di novo cancers separate from theoriginal cancer for which the host was treated using the autogenicvaccine.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a methodfor stimulating a directed immune response in cells of a livingorganism. More specifically, it is an object of the invention to providea means for stimulating immune response in cells of a mammal, andparticularly of a human, by isolation, separation, and propagation ofprecursor DCs in high yield from the blood and marrow of patientsafflicted with cancer. It is a further object of the invention that thismeans provide for predetermining the evolution of precursor cells intoprogrammable antigen presenting cells suitable for presentation bymixing to pre-selected antigens to elicit a host immune response capableof recognizing any or all expressed markers of tumorous cells or anyother “non-self” tumorous cells or RNA of said tumor cells, orexpression products encoded by such RNAs, or other non-self antigens.The invention further contemplates a process for aiding treatment ofboth early and terminal stages of cancer and other infections anddiseases.

A primary object of the invention is to provide a method of preventing,treating, reducing the severity, or possibly curing a disease in thesubject by stimulating the subject's immune response against thedisease. The scope of the invention further contemplates providingtreatment methodology for the whole spectrum of human diseases thesuccessful treatments of which rely on stimulation of the immune systemfor fighting infections and cancerous states.

Another object of the invention is using pAPCs in treating solid tumorsvia a protocol wherein pSAPCs derived from pAPCs act as vaccines. Yetother specific objects of the invention include adding cancer cell RNAand RNA expression products directly into a pAPC whereby the proteinantigens encoded by such RNA can be expressed on the surface of thepSAPC. Another embodiment of the invention contemplates extracting tumorRNA, cloning such RNA into bacterial expression vectors, thentransforming the pAPCs with said vectors so that tumor gene products maybe directly propagated in the pAPC for high level presentation by thepAPC to induce a specific immune response.

The invention further contemplates the creation of a vaccine to specificcancers including lung, prostrate, and breast cancers. Moreover, apreferred embodiment of the invention contemplates the creation ofallovaccines which can be created by donor DCs adhering to the ninebasic MHC-I and MHC-II phenotypes. Such allovaccines are furthercontemplated to include the immortalization of the nine DC lines knownto derive from precursor CD 34 stem cells utilizing currently wellcharacterized Epstein-Barr virus and other immortalization techniques(such as by retro and adenoviruses) understood by those skilled in theart. This embodiment contemplates that the immortalized allogenic DCswill be “loaded” with specific cancer cells, or RNA in toto or the polyA+ portion thereof, or gene products thereof, or bacterial expressionvectors containing tumor cell genes derived from said RNA thereof,followed by mixing the treated DCs with a host's plasmaphoresed T4and/or T8 cells which mixture or resegregated cells (T4, T8, or pSAPC)would then be returned to the host's system to induce the desiredlasting immune response to the specified antigens and cancer cellmarkers.

The present invention provides for a method of extracting and separatingprecursor DCS from the blood of a mammal. A preferred embodimentcontemplates extracting and separating a subject host presenting adiseased state the treatment of which requires host immunomodulation.The invention contemplates plasmaphoresing the blood to separate andisolate CD34+ and/or CD34− lineaged cells. In another preferredembodiment, bone marrow cells may be used in place of fresh whole blood.The isolated cells are then incubated with a specific regimen oftreatments heretofore not appreciated in the art to create anantigen-presenting DC. The steps of creating the antigen-presenting DCsutilize an in vitro process to exclusively yield activated dendriticcells which can then be presented with a host's disease state tumor cellor component parts or constructs thereof as previously described. TheDCs thus activated are termed pSAPCs.

The invention contemplates generation of both autogenous pAPCs andallogenic pAPCs. Allogenic pAPC vaccines are created using the nine DCphenotypes based on the known histocompatibility complex antigens (MHC Iand MHC II). Both autogenous and allogenic pAPCs may be “programmed” toany one or combination of such antigens. Programmed presenting cells(pSAPCs) are capable of eliciting “upgraded” cellular antitumorresponses in the host's T lymphocytes population via MHC class I and IIpathways acting in association with accessory and adhesion molecules,such as B7-1, B7-2 and ICAM-1. The endocytic activities of thesepathways are marked by a capacity to vigorously ingest fluid phase andwhole cell lysates by macropinocytosis, and in turn deliver ingestedsolutes to prelysosomal MHC class II-rich vesicles for subsequentpresentation as MHC class II surface molecules. The specific andchanneled use of the DCs according to the present invention avoids thenatural activity of the T lymphocyte's response to a tumor cell whichtreatment regiments known in the art presently utilize. In suchregiments, the T lymphocyte must locate the antigen directly from thehost's blood fluids or from the surface of the tumor itself. Thus, the Tlymphocyte has only the chance to respond to antigens it can find,without the aid of highly activated programmed DCs, allowing for thetumor cell to evade immune surveillance either due to poorimmunogenicity, or lack of the presence of a recognizable surfaceantigen. In contrast, the pAPC of the current invention are capable of“digesting” a tumor, or its RNA, or expression products of such RNA,selecting suitable antigens so derived from the tumor, and expressingthe antigens on the surface of the pAPC for presentation to other cellsof the immune system either in vitro or in vivo.

A preferred embodiment of the invention contemplates utilizing the pAPCsas effective autogenic and allogenic vaccines and boosters against tumorantigens and cell markers in vitro or in vivo. Moreover, since the pAPCshave the capacity to become immortalized, the current inventioncontemplates creating banks of immortalized cells comprising activatedpAPCs for use as allogenic vaccines and boosters.

The invention further contemplates mixing the pSAPCs with a host's CD4+Thelper cells and CD8+T killer cells which are isolated from whole bloodor bone marrow. Such mixing under conditions of the present inventionallows the antigens on the surface of the pSAPCs to be presented to theT cells, which presentation allows the T cells to become sensitized tothe cancer-specific antigens. It is well-known in the art that suchcancer-specific T lymphocytes are capable of mediating effective immunesurveillance against subsequent manifestation of the specific cancer.Thus, once “activated” as described herein, the CD4+ T helpers and CD8+T killer cells can be utilized as effective autogenic vaccines andboosters upon re-injection into the subject host, having been treatedfor activation according to the presently described and heretoforeunrecognized method.

The mechanism of stimulation of the T cell is believed to occuraccording to the following description. First, naive CD4+ T cells whenstimulated via presentation to the pAPC produced MHC class II antigens,become “educated” and initially produce IL-2. Next, they develop eitherinto educated TH1 or educated Th2 cells, depending on the specificregiment utilized to produce the pAPC including the nature of cytokineregulation utilized, type of antigen presenting cell used, and theexpression of accessory molecules of such cells. It is known that Th1cells produce IFN-γ, TNF-β and IL-2, while Th2 cells are known toproduce IL-4, IL-6, and IL-10. There are cross-regulatory effectsbetween Th1 and Th2 cells mediated by the cytokines in the form ofcross-cytokinic stimulation and inhibition. For instance, IL-4 inhibitsthe development of IFN-γ producing cells while dysregulated IL-10production normally serves to limit Th1 lymphocyte response.

The DC programming technique of the present invention uses the host'sdendritic cells derived from CD 34+ and CD34− cells to createprogrammable antigen-presenting DCs which are used in turn to stimulateactivation of cytotoxic T-4 helper lymphocytes. The T-4 helpers in turnactivate T-8 killer lymphocytes which attack tumor cells directly. TheDCs ingest fluid phase-nonbinding antigens from whole cell lysates, orsuch cells' RNA, or expression product of said RNA. The DCs may also betransformed with bacterial expression plasmids containing tumor cellcDNA. Once the DC has ingested said tumor antigens or has expressedtumor RNA, such antigens and expressed products are exocytosed afterbeing processed by prelysomal MHC-I and MHC-II vesicles. From theseprelysomal vesicles various antigens of the host's tumor cells aretransmitted to the surface of the now superpresenting pSAPC. The pSAPCselicit effective antitumoral responses by presenting antigens to T-celllymphocytes via MHC class I and MHC class II pathways as well asexpressing necessary accessory and adhesion molecules such as ICAM-1,B7-1, and B7-2.

Another preferred embodiment of the invention contemplates takingadvantage of the pAPC's prolific expression of adhesion and accessorysurface molecules and the responsiveness of T-lymphocytes to the pAPCpresented antigens which responsiveness is overpowering compared to thepreviously evasive detection experienced by other immune stimulationprotocols.

Yet another embodiment of the invention contemplates use of thesuccessful activation and population expansion of tumor-specific T-4 andT-8 lymphocytes (utilizing phoresed lymphocytes in vitro) to provide ameans of boosting old antergized T-cell populations.

As is understandable to those skilled in the art, the tumoral maskingand successful camouflaging experienced in past treatment protocols dueto anti-Th 1 measures of contra-IL-10, IFN-gamma, and TGF-β are overcomeby the pAPC of the present invention. Moreover, the present inventionmay also circumvent the temporary nature of enhanced solid tumorimmunogenicity obtained in gene insertion/deletion therapies ofIL-2/TGF-β caused by B7 and ICAM I or the interference from CD28/CTLA-4and LFA-1, or effects of B7 on induction of INF-gamma. By co-incubationof pAPCs with the host's tumor cells, with subsequent incubation withhost T cells, the resulting antigens displayed and immunogenic responseto be observed is profound.

The DC programming technique of the present invention furthercontemplates utilizing RNA from cancer cells that have already beenexcised from patients and either maintained in frozen preservation orpreserved in paraffin cell blocks used for histology analysis. Thus,with this invention, one need only take surgical tumor specimens byinvasive means on one occasion. The material preserved in paraffinblocks may be de-paraffinated by standard laboratory techniques and themessenger RNA from the tumor cells extracted. Whether from frozen stockor paraffin block, this RNA, which includes the cell's mRNA, may then beused for direct incorporation into the dendritic cells or, may be usedthrough standard in vitro expression methods to obtain encoded mRNAexpression products for direct incorporation into the DC, or may bereverse transcribed into cDNA and cloned into bacterial expressionvectors for transformation of the DCs.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects or features and advantages of theinvention will be made apparent from the following detailed descriptionof the preferred embodiments of the invention and from the drawings inwhich:

FIG. 1 is a schematic chart indicating the cell differentiation lineageof hematopoietic cells indicating the evolution channel of stem cells;

FIG. 2 is a graph showing results of proliferation assay on “loaded” Tlymphocytes of human DCs programmed and grown from base stock ofautologous CD34+HPC from peripheral blood, cultured with GM-CSF, IL-4and TNF-_(χ);

FIG. 3 is a pictorial chart delineating progressive steps to be taken instimulating CD34 cells to create antigen-presenting DCs; and

FIG. 4 is a pictorial chart delineating basic steps to create programmedpAPCs, activated T cells, and the subsequent methods of administrationof such pAPC/T cell mixture to a subject host.

DETAILED DESCRIPTION

It will be appreciated by those skilled in the art that immunogenicpotential exists in most human blast cells and that precursor Blymphoblasts present tumor and MHC-associated antigens. Thispresentation is well-known to elicit specific T lymphocyte response tosuch antigens. A primary embodiment of the current invention rests onthe inescapable conclusion to be drawn from the T lymphocyte response toB lymphoblast antigen presentation that human dendritic cells if primedby co-culturing them with autologous lymphoblasts, or whole cell lysatestherefrom, or their RNA in toto, or the poly A+ portion thereof, orexpression products of such RNA therefrom, or are transformed withexpression vectors containing cDNA thereof, will also act in presentingantigen to T lymphocytes. Recent strategies to stimulate the developmentof tumor specific T lymphocyte responses have utilized professionalantigen presenting cells, including dendritic cells, to elicit effectivecellular antitumor responses by presenting antigens to T lymphocytes.This presentation and response is attributable to the professionalantigen presenting cell's ability to process antigens via the MHC classI and II pathways in association with accessory and adhesion molecules,including B-7, B7-21 and ICAM-1 which are necessary to ensure a Tlymphocyte response for the reason that tumor cell immunogenicity isoften poor. In the current invention, DCs are utilized for their antigenpresenting properties and ability to prime naive CD4T-helper cells viaMHC class I and II CD8+ cytotoxic T lymphocytes (CTL) directly. DCs havethe ability to vigorously ingest nonbinding antigens, such as whole celllysates, and further have the ability to deliver bits and pieces of suchlysates, or their RNA in toto, or transcribed poly A+, or proteinsencoded by such RNA, to MHC class II-rich vesicles for incorporationinto peptides prior to display on cell membranes as MHC class II surfacemolecules.

The mechanism of the current invention differs from prior vaccinepreparation processes in that DCs of the present protocol are primedwith lymphoblasts prepared as either whole cell lysates, isolated RNA,expressed RNA gene product, or expression vector containing reversetranscribed mRNA, rather than a derived lymphokine peptide and/or wholeintact tumor cell. This approach is taken for two reasons. First,expression of individual antigen markers in prior research has provenineffective. Second, the use of autologous tumor cell lysates willprovide simultaneous immunization against all markers present in thehost's tumors, and thus avoid selection of resistant lymphoblastantigen-loss variants. Moreover, not only does the whole cell lysatemethod avoid antergy, it also avoids the masking effect that some tumorsexhibit. In a preferred embodiment, this invention focuses on primingDCs at an early stage in their maturation cycle when antigen captureexhibits greatest potential. Recognition of this aspect of cell responseactivity is an important part of the success experienced by thisinvention. Thus, the methodology of the current invention for elicitingresponse in lymphoblasts insures a “shotgun” response. In contrast,prior research has centered on singly identified antigenic moietieswhich have had the potential of responding to only one type of antigenbeing “expressed” while missing other surface expression molecules. Theprior research therefore provides for selection of only a subpopulationof tumor cells while missing others. Understandably, recurrence of tumortissues may occur within a few months. This is why gene therapy issuccessful at first but subsequently shows disappointing results afterseveral months.

According to the preferred objects of the invention, and referringgenerally to FIGS. 1, 3 and 4, the steps necessary to direct CD34+ andCD34− stem cell to become a super antigen presenting cell include thefollowing general outline. (First, FIG. 1 shows the cell differentiationlineage of hematopoietic cells. The protocol of the present inventiondirects CD 34+ and CD34− cells to antigen-presenting dendritic cells.Peripheral whole blood or bone marrow cells are cultured in the presenceof SCF, GM-CSF, IL-4, and TNF-_(χ). The resulting cell populationcomprises antigen presenting DCs which are programmable with any antigenengulfed by them. These cells may also be referred to as pAPCs. Thecells are then “loaded” or “programmed” using irradiated whole celllysates, cell RNA or the poly A+ portion thereof, or expression productsof cell RNA or transcribed RNA, or cDNA of cell mRNA, or plasmidexpression vectors containing such cDNA, of autologous lymphoblasts.Programmed DCs are used as T lymphocyte stimulator cells while a smallportion is reserved in cytopreservation. After addition of the Tlymphocytes in the presence of TNF-_(χ), proliferation of T lymphocytesis then measured by 3H-thymidine uptake. As shown in FIG. 2, the Tlymphocytes are dramatically stimulated to proliferate.

In the aforementioned protocol, stem cell factor is used to expand thepool of hematopoietic progenitor cells, GM-CSF is utilized to promotethe growth and maturation of dendritic cells, IL-4 is used to suppressovergrowth by macrophages, and TNF-_(χ), is added after about 5 days inculture to facilitate maturation of DCs. The culture is then propagatedto create an autologous vaccine comprising antigen presenting dendriticcells.

EXAMPLE I (CD34+)

The preparation of pSAPCs by directing the maturation of CD34+ cells issimilar to the methodology used to direct CD34− cells. However there aredistinct protocol differences. For CD34+ directed maturation hostperipheral blood is ficolled, washed and resuspended in PBS/0.1% BSA.The cell suspension, consisting of “adherent” (monocytes andmacrophages) and “nonadherent” (stem cells, T4, T8 and B cells) areseparated such that the nonadherent cell types are retained. Thisnonadherent mixture is then reacted with Dynal magnetic beads (indirect)which are sensitive for T4, T8 and B cells. The stem cells from whichpAPCs will be generated are left in solution. The beads are thencentrifuged and cells in solution are stained using directly conjugatedFITC and PE-labeled CD3, CD 19, CD34, CD 14, CD45. The stained cellsuspension is then analyzed by flow cytometry. Desired fractions arecollected and cultured in AIM 5 (made by Gibco) in 10% FBS, 10% HS, 1%NEAA, 1% NaPyruvate, 1 Pen/Strep+ 800 U/ml GM-CSF, 500 U/ml IL-4, and 20mg/ml SCF at 37° C. in 5% C02. The cells are cultured for 4 to 6 daysaccording to standard suspension culture protocols. Cytopreparations areobtained and stained with Wright's/Giemsa for flow cytometric analysisevery four days during culture. The cells are examined for thefollowing: CD2, CD 13, CD 14, CD 19, CD34, CD45, CD80, CD86, CD1a, HLA-1and HLA-DR. The dendritic cells are then pulsed with any one orcombination of the following: 1) “foreign” (tumor) cell RNA in toto, 2)foreign poly A+ RNA, 3) expression product of foreign mRNA, 4)expression plasmid containing cDNA from tumor cells, after which growthmedia is added for a concentration of 2×10⁶ cells/ml for 72 hourcoculture at a concentration equivalent to a 1:1 lymphoblast todendritic cell ratio. These loaded dendritic cells are thencryopreserved or used immediately as T lymphocyte activation cells.

Where whole tumor lysate tissue is to be added to the DCs, the lysate istreated with standard rapid freeze thaw method then irradiated with upto 40 Gy gamma source to insure that there is no viable cell or nucleicacid. Otherwise the RNA isolated by standard methodologies and eitherused in toto or fractionated to isolate the poly A+ fraction. The RNA,either in toto or as poly A+, is then either added directly to the DCs,translated using standard in vitro methodologies and the expressionproducts with the RNA added to the DCs, or is reverse transcribed intocDNA, cloned into an expression vector and then transformed into the DCsby standard techniques. Regardless of the treatment of the RNA or celllysate, such material is presented to the pAPCs approximately 4 to 7days after initial culture of the DCs. The pAPCs are retreated withlysate or RNA derived material as above described again at about day 10and at about day 15 from initial culture of the dendritic cells.

Following maturation of the cells as described in the above steps, T4helper leukocytes and T8 killer leukocytes are added to the pSAPC/tumorcell culture and allowed to incubate for about 5 days. Importantly, themorphology of the T cell pool is examined to ensure that they are not ina presently excited or activated state. Such state is recognizable bythe increased size normally associated with activated state T4 and T8cells. If the morphology indicates that the cells are activated, theyare first allowed to return to an ambient state prior to introduction tothe DCs. The T4/T8 pool is added at about a 1:1 ratio of T4/T8 to pAPC.Following this incubation period, the cell culture is again subjected tofull flow cytometry analysis for separation and isolation of the T4 andT8 cells. Depending upon the proliferation count, the T cell pool and/ordendritic cell populations may be introduced into the host. Where the Tcell count has not reached satisfactory levels, the cells may beintroduced to the pSAPCs for further culture. Moreover, the T cells maybe reintroduced where T cell/dendritic cell populations have reachedsatisfactory levels for the purpose of hypersensitizing them to thedesired antigens. Where restimulation of T cells by pSAPC iscontemplated, it is preferred to allow the educated T cell pool toreturn to an ambient state. Besides adding the T cell pool to the hostalone, these cells may be introduced to the host in conjunction with thepSAPC. Moreover, the pSAPCs may be introduced to the host alone. Whetherintroduction to the host is by T cells alone, T cells plus pAPCs, orpAPCs alone, the fraction(s) are presented to the host system foreliciting sustained immune response (i.e., memory).

EXAMPLE II (CD34−)

The ability of utilizing the potential of CD34− cells provides formarked improvement to current methodologies known in the art. It isknown that peripheral blood contains up to approximately a ten to oneratio of CD34− to CD34+ cells. Use of CD34− cells provides for a vastincrease in cell population that can be effectively utilized in cancertherapy regimens. Moreover, the difficulties associated withleukophoresis procedure currently carried out to obtain sufficientquantities of stem cells will be avoided. Currently CD34− cells arediscarded as it is believed that they are precommitted to becomemacrophages. However, CD34− cells may be programmed towards dendriticcommitment with proper incubation and lymphokine stimulation.

In this example, the cell donor is pretreated with IL-3 and either G-CSFor GM-CSF. For adult patient/donors, the regimen is administration ofIL-3 at 15–55 ug/Kg/day for 4 consecutive days followed by 3 dayswithout such administration. Next, either G-CSF or GM-CSF isadministered at 15–55 ug/Kg/day for 5 days followed by 2 days of bloodretrieval from the donor/patient of 50 to 90 ml of venous blood drawneach day. Blood is drawn in heparinized syringes. Such pretreatment mayyield a higher number of CD34+ and CD34− cells up to 40×multiple ofprogenitor cells over that normally found in peripheral blood. After thepretreatment period, blood is removed from the donor and the white bloodcells are removed by standard protocol. Following magnetic beadseparation as described above in example 1, the stem cells containingCD34+ and CD34− cell populations are washed in PBS containing 1% humanautologous plasma four times by standard laboratory techniques. Thecells are then cultured in RPMI 1640 culture medium containing 1.0%heparinized autologous human plasma, 10 mM Hepes, 20 ug/ml Gentamicine,1×10⁵ U/ug rhGM-CSF (Kirin), and 0.5×10⁵ U/ug rhIL-4 (Immunex). Theculture is allowed to incubate in the above media for 7 days at 37° C.during which the cells are replenished every other day with fresh mediaand lymphokines. This process of in vitro incubation allows the CD34−cell population to become immature dendritic cells. If cell populationshave not reached adequate levels during this 7 day incubation, they maybe allowed to grow an additional 7 days using the same growth mediaprotocol. Moreover, following this protocol, the cells may be preservedin frozen culture at this stage.

After the above growth cycle, the non-adherent cells are collected bypouring off the growth media via moderately vigorous aspiration andtransferred to fresh 6 well IG coated plates (Bacteriologic platesFalcon) in the following protocol. The plates are first washed with PBSfollowed by addition of 4 ml/plate of human gamma-globulin (ChappelLabs) at a concentration of 10 mg/ml for 1 minute. The plates are thenwashed 3 times in PBS. The stem cell population from above growth cycleare added to the IG plates at a concentration of 5×10⁷ cells per well ina volume of 6 to 8 ml for 1 hour at room temperature. Following thisincubation, the plates are washed to remove non-adherent cells by gentleaspiration and the remaining adherent cells are incubated for 3 days at37° C. in medium containing 1.0% autologous plasma and SACS (fixedstaphylococcus aureus 1/10,000 dilution), Pansorbin (Calbiochem) at adilution of 1/10,000, 1640 RPMI, 10 mg/ml Hepes, and 20 ug/mlGentamicine. From this cell mixture the immature CD34− cells areconditioned to become mature dendritic cells expressing among otherreceptors CD83 which is a co-receptor to T cell activation.

After the 3 day maturation cycle, the mature dendritic population isready to be pulsed with tumor antigenic materials as described inexample 1 to become pSAPCs followed by use in activating T and B cellpopulations.

A constant pitfall encountered in prior attempts at creating a dendriticvaccine was the dependence on the circulation in the peripheral blood ofspecific T lymphocytes generated in central lymphoid organs after thevaccine has been administered. This problem has been overcome by our“preprogramming” of precursor dendritics. Therefore, “traveling” throughthe body becomes programmed-in under our maturation process. Although acomplete host immune response is anticipated, T4 or T8 response could besubnormal in some individuals as assayed by flow cytometry analysis. Tocover this contingency, a small portion of phoresed blood components areset aside initially as a reserve in case initial treatment assays arebelow “par”. Par is determined by standard multiparameter flow cytometrywith monoclonal antibodies and fluorescent reagents. These assays atbaseline and 30, 60, 90 and 120 days use standard cytotoxic T-lymphocytelimiting dilution assays. An additional reserve is set aside before thevaccine is infused into the host for the purpose of providing a sourceof booster material to be used at 30 and 60 days after initial infusion.These time intervals are chosen due to the production cycle of pAPCsbeing 21 days. Since the host's original irradiated tumor cells arephenotypically identified, the fact that they may not be clonogenic doesnot affect the host's pAPCs from expressing homologous associatedeffective MHC I and II class surface antigens.

It will be appreciated by those skilled in the art that the aboveprotocol may be used with any of the known cancer or tumor diseasestates to detect, and propagate an immune response to antigens,receptors, ligands or other cellular components expressed by such cancerand tumor cells. Examples of cancer cell types and the identifieddiseases contemplated for use with the embodiments of the inventioninclude: 1) that spectrum of disease states characterized by dysfunctionof the T cell system such as psoriasis, seborrheic dermatitis and otherskin diseases: 2) T cell lymphoma; 3) systemic acute autoimmune diseasestates such as rheumatoid arthritis and systemic lupus erythematosis; 4)organ specific autoimmune states such as Crohn's disease, multiplesclerosis, Guillain Barre′ syndrome, Graves disease, pernicious anemia,Addison's disease and sarcoidosis; 5) HIV/AIDS wherein pAPC is targetedto infected T4 cells; 6) specific carcinomas such as adenocarcinoma ofprostate, breast, colon, ovary, uterus, stomach, kidney and pancreas; 7)lung carcinoma of all histological types such as adenocarcinoma,squamous cell carcinoma, small cell carcinoma, large cell anaplasticcarcinoma; 8) malignant brain tumors such as astrocytoma andglioblastoma multiforme; 9) urinary tract carcinoma includingtransitional cell, squamous cell, and adenocarcinoma; 10) squamous cellcarcinoma of the esophagus; 11) infections such as bacterial, viral, andfungal.

Modifications and other embodiments of the invention will be apparent tothose skilled in the art to which this invention relates having thebenefit of the foregoing teachings, descriptions, and associateddrawings. The present invention is therefore not to be limited to thespecific embodiments disclosed but is to include modifications and otherembodiments which are within the scope of the appended claims.

1. A method of stimulating a disease-specific immune response, themethod comprising infusing an immunogenic cell composition capable ofstimulating a disease-specific immune response into a patient whereby adisease-specific immune response is initiated, the immunogenic cellcomposition being prepared by: a. isolating hematopoietic stem cellsfrom a donor; b. incubating the stem cells in a cell culture mediumcomprising GM-CSF, SCF and IL-4, whereby the stem cells are directed todevelop into programmable antigen presenting cells (pAPCs); c.incubating the pAPCs in an incubation vessel coated with humanγ-globulin, whereby the pAPCs become adherent; d. removing non-adherentcells from the incubation vessel; e. further incubating the adherentpAPCs in a cell culture medium comprising autologous human plasma and afixed staphylococcus aureus solution; f. mixing the pAPCs with cellularmaterial derived from cells positive for the disease, the cellularmaterial selected from the group consisting of cell lysate, cell RNA,cell poly A+RNA, cDNA derived from cell RNA, and cDNA derived from cellRNA incorporated into an expression vector; g. incubating the pAPCs withthe cellular material, whereby loaded pAPCs are produced; and h.incubating the pAPCs with TNF-a after the pAPCs have been loaded,whereby disease-specific programmed super antigen presenting cells(pSAPCs) capable of stimulating an immune response to the disease areproduced.
 2. The method of claim 1 where the donor and the patient areone in the same.
 3. The method of claim 1 where the donor and thepatient are not one in the same.
 4. The method of claim 1 wherein thehematopoietic stem cells are selected from the group consisting of: a.CD34 positive hematopoietic stem cells; and b. CD34 negativehematopoietic stern cells.
 5. The method of claim 4 wherein the donor issubject to a pretreatment protocol with agents that increase the numberof hematopoietic stern cells in the donor's peripheral blood, thepre-treatment protocol selected from the group consisting of: a. IL-3treatment for 4 consecutive days, followed by 3 days of no treatment,followed by GM-CSF treatment for 5 consecutive days; b. and IL-3treatment for 4 consecutive days, followed by 3 days of no treatment,followed by G-CSF treatment for 5 consecutive days.
 6. The method ofclaim 5 where the donor and the patient are one in the same.
 7. Themethod of claim 5 where the donor and the patient are not one in thesame.
 8. The method of claim 1 further comprising incubating the pSAPCswith a T-lymphocyte preparation comprising a plurality of T-lymphocyteswhereby activated disease-specific T-lymphocytes are produced.
 9. Themethod of claim 8 where the plurality of T-lymphocytes in theT-lymphocyte preparation is selected from the group consisting of: a.CD4 positive T-lymphocytes alone; b. CD8 positive T-lymphocytes alone;c. both CD4 positive and CD8 positive T-lymphocytes; and d. unsegregatedT-lymphocytes.
 10. The method of claim 8 where the donor and the patientare one in the same.
 11. The method of claim 8 where the donor and thepatient are not one in the same.
 12. The method of claim 8 wherein thehematopoietic stem cells are selected from the group consisting of; a.CD34 positive hematopoietic stem cells; and b. CD34 negativehematopoietic stem cells.
 13. The method of claim 12 wherein the donoris subject to a pretreatment protocol with agents that increase thenumber of hematopoietic stem cells in the donor's peripheral blood, thepre-treatment protocol selected from the group consisting of: a. IL-3treatment for 4 consecutive days, followed by 3 days of no treatment,followed by GM-CSF treatment for 5 consecutive days; b. and IL-3treatment for 4 consecutive days, followed by 3 days of no treatment,followed by G-CSF treatment for 5 consecutive days.
 14. The method ofclaim 13 where the donor and the patient are one in the same.
 15. Themethod of claim 13 where the donor and the patient are not one in thesame.